The 2005 Nobel Prize in medicine/physiology was awarded to the two scientists who discovered the Helicobacter pylori (Hp). Helicobacter pylori is which is believed to be an important pathogenic bacterium for diseases of upper digestive tract in human and the main pathogenic cause of chronic gastritis, gastric ulcer, and duodenal ulcer. Helicobacter pylori World health organization (WHO) has also confirmed that Hp is a pathogenic bacterium closely related to gastric cancer, classifying it as the primary carcinogenic factor. Hp is one of the bacteria with the highest infection rate in the world, with an infection rate reaching 50% in the global population and even higher in developing countries. In China, there are as many as six hundred million people infected with Hp, and every year two hundred thousand people die of gastric cancer. Thus, Hp is a major threat to human health. Despite of positive clinical significance, today's antibiotic therapy of Hp infection still has some obvious drawbacks: 1) toxic and adverse side effects; 2) generation of drug resistant strains; 3) high cost; 4) lengthy treatment cycle and poor patient compliance; and 5) unstable therapeutic effect. On the other hand, vaccine is considered the most cost-effective means to control infectious diseases. Through inducing a specific immune response in the body, vaccination can be used to prevent or treat Hp infection. While the research progress in whole-cell vaccines is limited by the difficulty in large-scale cultivation of Hp and the existence of potential carcinogen in crude antigen, the development in genetically engineered vaccines has become the main direction owing to its safety, effectiveness, low cost, and ease in promotion and use. Although intensively researched both domestically and abroad, Hp vaccines have yet to be successful.
Urease, a nickel-dependent enzyme, catalyzes hydrolysis of urea into ammonia and carbonic acid, and is the mostly abundant protein expressed in Helicobacter pylori both inside cells and on cell membrane, accounting for 5%-10% of the total Hp protein. Urease decomposes urea to produce ammonia, helping bacteria to colonize in the stomach by neutralizing gastric acid as well as supplying ammonia for bacteria protein synthesis. Therefore, Host tissues of Hp may be injured directly by ammonia or indirectly by the stimulation of urease-induced inflammatory reaction. Blockage of urease gene expression can inhibit the colonization of Helicobacter pylori in the host, reduce bacterial protein synthesis, and lower the Helicobacter pylori-related inflammatory reaction. Urease B antibody can be detected in all Hp-infected patients especially those with notable symptoms, and the antibody level is, to certain extent, relevant to the seriousness of disease conditions. Oral injection of Helicobacter pylori urease or recombinant urease B subunit (rUreB) can protect mice against Helicobacter pylori infection and eliminate preexisting infection (Michetti et al., Gastroenterol. 1994). The activity of urease seems to play an important role in Hp infection, as Hp with loss of activity of urease does not result in infection in animal model. Therefore, an antibody that neutralizes the activity of urease may play a crucial role in resisting against Hp colonization. The above findings indicate that urease antibodies, especially one that is capable of neutralizing the activity of urease, could exert the main role in resisting against Hp infection.
Mucosal immune system is an important part of the body's immune system, mainly including intestinal mucosa-associated lymphoid tissue and bronchus associated lymphoid tissue etc., and it exerts unique action with respect to body defense function. Mesenteric lymph nodes and large quantity of lymphocytes dispersed in lamina propria mucosae and intestinal epithelia constitute immune induction sites and immune effect sites. Through uptaking, processing and extracting antigens, various immunocytes on the surface of gastrointestinal mucosa etc. produce and secrete antigen-specific antibodies (mainly sIgA) which specifically bind with antigen-carrying bacterial or viral vectors to prevent them from colonizing to mucosa surface or invading body, thus exerting certain immunological defense function.
However; the severest defect of mucosal immune system is that it is liable to develop immunological tolerance to antigens. Even with enhanced level of antigen, the level of antigen-specific sIgA produced by the body or mucosa remains very low, and the immune defense ability to the infection of the antigen-carrying microorganisms is dismal.
Heat labile toxin (LT) is a heat-labile enterotoxin produced by enterotoxigenic Escherichia coli (ETEC) and capable of inducing severe diarrhea in human and some domestic animals. LT is composed of one A subunit (LTA) and five B subunits (LTB). Spatially five completely identical LTBs form a circular pentamer, and the center of which lies the LTA with its C terminal bound to LTB via noncovalent bonds. The A subunit is composed of two subunits A1 and A2, linked by disulfide bond, wherein A1 is the toxic portion of the toxin whereas A2 binds with the B subunit. All the A and B subunits in cytoplasm exist in the form of precursor carrying signal peptides, and only assemble into intact LT after passing through cell membrane. The B subunit serves to specifically bind with GM1-ganglioside receptor on the surface of eukaryotic cell to make the conformational transition of LT molecule; the A subunit dissociates from the B subunit and enters into cell membrane, followed by disulfide bond degradation and A1 peptide chain activation; and the A1 subunit has the activity of GTP-independent ADP-ribosylation transferase, which destructs the degradation and balance of intracellular cAMP through G protein-mediated ADP-ribosylation reaction and triggers the rise of cAMP level, thus inducing toxin effect.
LT is considered a promising mucosal immune adjuvant in recent years. The research by Rollwagen etc. showed that LT can strengthen mucosal immune response to campylobacteria and accelerate the elimination of the bacteria in the intestinal tract; and furthermore, animals initially immunized with LT and an antigen do not develop immunological tolerance to the antigen even after a long-term observation.
The viewpoint that LT can be used as mucosal immune adjuvant has been generally accepted (Lycke N, et al. Immunology, 1986, 59(2):301-308; Clements J D, et al. Vaccine, 1988, 6(3):269-277; Giuliani M M, et al. J. Exp. Med., 1998, 187(7):1123-1132). Since LT has very strong intestinal toxicity, consequently its B subunit or constructed nontoxic or low toxic LT mutant were mainly used as adjuvant (Yamamoto M, et al. J. Immunol., 1999, 162:7015-7021; Martin M, et al. J. Immunol., 2002, 169(4):1744-1752; Tamura S I, et al. Vaccine, 1994, 12:1238-1240). However, the A subunit of LT, in addition to its ADP-ribosyl enzyme activity, also relates to adjuvant activity (Giuliani M M, et al. J. Exp. Med., 1998). LTA chain stimulates the isotype switching of mucosa-associated B cells and meanwhile LTA participates in the initial phase of mucosal immune response, making type-fixed IgA B cells migrate from production site to distal effect site (DeHann L, et al. Vaccine, 1996; (4):260-266). De Haan etc. found that LT mutant lack of ADP-ribosyl enzyme activity still possesses adjuvant activity, suggesting that the adjuvant activity of LT is independent of the A1 subunit with ADP-ribosyl enzyme activity while the A2 subunit may contribute to the adjuvant activity of LT. De Haan's experiments also demonstrated that, through immunizing mice via nasal cavity mucosa, the LT mutant without toxic activity still keeps the immune properties of the wild-type toxin The single use of recombinant LTB shows a weaker immunogenicity than that of LT mutant without toxic activity (De Haan L, et al. Infect Immun, 1996), suggesting that the ADP-ribosyl enzyme activity of LTA does not directly relate to the immunity of the toxin. Additionally, LT whole toxin produces high-level systemic IgG and mucosal S-IgA responses while equal amount of LTB merely produces a low-level IgG response. That the immunogenicity of LT is stronger than that of LTB indicates that LTA plays an important role in the immune response of the toxin. Whereas the GTP-dependent ADP-ribosyl transferase activity possessed by the A1 subunit is related to the toxin effect, the adjuvant effect of the LTA is hinted mainly exerted by LTA2 subunit.
LTB has been used as an immunological adjuvant at present. For example, LTB is fused with UreB to form recombinant protein LTB-UreB (Wuchao, Zou quanming etc. Research on fusion and expression of Helicobacter pylori UreB and Escherichia coli LTB genes. Chinese Journal of Microbiology and Immunology, 2002, 22(2):175-179), and can be prepared into intramolecular adjuvant vaccine. However, the immunoprotection rate of the recombinant protein has yet to reach the desired level.
Based on above-mentioned, a recombinant Hp subunit intramolecular adjuvant vaccine with enhanced strength and complete protection against Helicobacter pylori infection, that can also be conveniently administered, is especially of demand in the field.